The present invention provides a method of synthesising oligonucleotides and oligonucleotide phosphorothioates in solution based on H-phosphonate coupling and in situ sulfur transfer, carried out at low temperature. The invention further provides a process for the stepwise synthesis of oligonucleotides and oligonucleotide phosphorothioates in which one nucleoside residue is added at a time, and the block synthesis of oligonucleotides and oligonucleotide phosphorothioates in which two or more nucleotide residues are added at a time.
In the past 15 years or so, enormous progress has been made in the development of the synthesis of oligodeoxyribonucleotides (DNA sequences), oligoribonucleotides (RNA sequences) and their analogues xe2x80x98Methods in Molecular Biology, Vol. 20, Protocol for Oligonucleotides and Analogsxe2x80x99, Agrawal, S. Ed., Humana Press, Totowa, 1993. Much of the work has been carried out on a micromolar or even smaller scale, and automated solid phase synthesis involving monomeric phosphoramidite building blocks Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862 has proved to be the most convenient approach. Indeed, high molecular weight DNA and relatively high molecular weight RNA sequences can now be prepared routinely with commercially available synthesisers. These synthetic oligonucleotides have met a number of crucial needs in biology and biotechnology.
Following Zamecnik and Stephenson""s seminal discovery that a synthetic oligonucleotide could selectively inhibit gene expression in Rous sarcoma virus, (Zamecnik, P.; Stephenson, M. Proc. Natl. Acad. Sci. USA 1978, 75, 280-284), the idea that synthetic oligonucleotides or their analogues might well find application in chemotherapy has attracted a great deal of attention both in academic and industrial laboratories. For example, the possible use of oligonucleotides and their phosphorothioate analogues in chemotherapy has been highlighted in the report of Gura, T. Science, 1995, 270, 575-577. The so-called antisense and antigene approaches to chemotherapy (Oligonucleotides. Antisense Inhibitors of Gene Expression, Cohen. J. S., Ed., Macmillan, Basingstoke 1989 Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-649), have profoundly affected the requirements for synthetic oligonucleotides. Whereas milligram quantities have generally sufficed for molecular biological purposes, gram to greater than 100 gram quantities are required for clinical trials. Several oligonucleotide analogues that are potential antisense drugs are now in advanced clinical trials. If, as seems likely in the very near future, one of these sequences becomes approved, say, for the treatment of AIDS or a form of cancer, kilogram or more probably multikilogram quantities of a specific sequence or sequences will be required.
In the past few years, a great deal of work has been carried out on the scaling-up of oligonucleotide synthesis. Virtually all of this work has involved building larger and larger synthesisers and the same phosphoramidite chemistry on a solid support. The applicant is unaware of any recent improvement in the methodology of the phosphotriester approach to oligonucleotide synthesis in solution, which makes it more suitable for large- and even moderate-scale synthetic work than solid phase synthesis.
The main advantages that solid phase has over solution synthesis are (i) that it is much faster, (ii) that coupling yields are generally higher, (iii) that it is easily automated and (iv) that it is completely flexible with respect to sequence. Thus solid phase synthesis is particularly useful if relatively small quantities of a large number of oligonucleotides sequences are required for, say, combinatorial purposes. However, if a particular sequence of moderate size has been identified and approved as a drug and kilogram quantities are required, speed and flexibility become relatively unimportant, and synthesis in solution is likely to be highly advantageous. Solution synthesis also has the advantage over solid phase synthesis in that block coupling (i.e. the addition of two or more nucleotide residues at a time) is more feasible and scaling-up to any level is unlikely to present a problem. It is much easier and certainly much cheaper to increase the size of a reaction vessel than it is to produce larger and larger automatic synthesisers.
In the past, oligonucleotide synthesis in solution has been carried out mainly by the conventional phosphotriester approach that was developed in the 1970s (Reese, C. B., Tetrahedron 1978, 34, 3143-3179; Kaplan, B. E.; Itakura, K. in xe2x80x98Synthesis and Applications of DNA and RNAxe2x80x99, Narang, S. A., Ed., Academic Press, Orlando, 1987, pp. 9-45). This approach can also be used in solid phase synthesis but coupling reactions are somewhat faster and coupling yields are somewhat greater when phosphoramidite monomers are used. This is why automated solid phase synthesis has been based largely on the use of phosphoramidite building blocks; it is perhaps also why workers requiring relatively large quantities of synthetic oligonucleotides have decided to attempt the scaling-up of phosphoramidite-based solid phase synthesis.
Three main methods, namely the phosphotriester (Reese, Tetrahedron, 1978), phosphoramidite (Beaucage, S. L. in Methods in Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 33-61) and H-phosphonate (Froehler, B. C. in Methods in Molecular Biology, Vol. 20, Agrawal, S., Ed., Humana Press, Totowa, 1993, pp 63-80; see also WO94/15946 and Dreef, C. E. in Rec. Trav. Chim. Pays-Bas, 1987, 106, p512) approaches have proved to be effective for the chemical synthesis of oligonucleotides. While the phosphotriester approach has been used most widely for synthesis in solution, the phosphoramidite and H-phosphonate approaches have been used almost exclusively in solid phase synthesis.
Two distinct synthetic strategies have been applied to the phosphotriester approach in solution.
Perhaps the most widely used strategy for the synthesis of oligodeoxyribonucleotides in solution involves a coupling reaction between a protected nucleoside or oligonucleotide 3xe2x80x2-(2-chlorophenyl)phosphate (Chattopadhyaya, J. B.; Reese, C. B. Nucleic Acids Res., 1980, 8, 2039-2054) and a protected nucleoside or oligonucleotide with a free 5xe2x80x2-hydroxy function to give a phosphotriester. A coupling agent such as 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-1H-triazole (MSNT) (Reese, C. B.; Titmas, R. C.; Yau, L. Tetrahedron Lett., 1978, 2727-2730) is required. This strategy has also been used in the synthesis of phosphorothioate analogues. Coupling is then effected in the same way between a protected nucleoside or oligonucleotide 3xe2x80x2-S-(2-cyanoethyl or, for example, 4nitrobenzyl)phosphorothioate (Liu, X.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1, 1995, 1685-1695) and a protected nucleoside or oligonucleotide with a free 5xe2x80x2-hydroxy function. The main disadvantages of this conventional phosphotriester approach are that some concomitant 5xe2x80x2-sulfonation of the second component occurs (Reese, C. B.; Zhang, P.-Z. J. Chem. Soc., Perkin Trans. 1, 1995, 2291-2301) and that coupling reactions generally proceed relatively slowly. The sulfonation side-reaction both leads to lower yields and impedes the purification of the desired products.
The second strategy for the synthesis of oligodeoxyribonucleotides in solution involves the use of a bifunctional reagent derived from an aryl (usually 2-chlorophenyl) phosphorodichloridate and two molecular equivalents of an additive such as 1-hydroxybenzotriazole (van der Marel, et al, Tetrahedron Lett., 1981, 22, 3887-3890). A related bifunctional reagent, derived from 2,5-dichlorophenyl phosphorodichloridothioate (Scheme 1b), has similarly been used (Kemal, O et al, J. Chem. Soc., Chem. Commun., 1983, 591-593) in the preparation of oligonucleotide phosphorothioates.
The main disadvantages of the second strategy result directly from the involvement of a bifunctional reagent. Thus the possibility exists of symmetrical coupling products being formed, and the presence of small quantities of moisture can lead to a significant diminution in coupling yields.
It is an objective of certain aspects of the present invention to provide a new coupling procedure for the synthesis of oligonucleotides in solution that in many embodiments (a) is extremely efficient and does not lead to side-reactions, (b) proceeds relatively rapidly, and (c) is equally suitable for the preparation of oligonucleotides, their phosphorothioate analogues and chimeric oligonucleotides containing both phosphodiester and phosphorothioate diester internucleotide linkages.
According to a first aspect of the present invention, there is provided a process for the preparation of a phosphorothioate triester which comprises the solution phase coupling of an H-phosphonate with an alcohol in the presence of a coupling agent thereby to form an H-phosphonate diester and, in situ, reacting the H-phosphonate diester with a sulfur transfer agent to produce a phosphorothioate triester.
The H-phosphonate employed in the process of the present invention is advantageously a protected nucleoside or oligonucleotide H-phosphonate, preferably comprising a 5xe2x80x2 or a 3xe2x80x2 H-phosphonate function, particularly preferably a 3xe2x80x2 H-phosphonate function. Preferred nucleosides are 2xe2x80x2-deoxyribonucleosides and ribonucleosides; preferred oligonucleotides are oligodeoxyribonucleotides and oligoribonucleotides.
When the H-phosphonate building block is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a 3xe2x80x2 H-phosphonate function, the 5xe2x80x2 hydroxy function is advantageously protected by a suitable protecting group. Examples of such suitable protecting groups include acid labile protecting groups, particularly trityl and substituted trityl groups such as dimethoxytrityl and 9-phenylxanthen-9-yl groups; and base labile-protecting groups such as FMOC.
When the H-phosphonate building block is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a 5xe2x80x2 H-phosphonate function, the 3xe2x80x2 hydroxy function is advantageously protected by a suitable protecting group. Suitable protecting groups include those disclosed above for the protection of the 5xe2x80x2 hydroxy functions of 3xe2x80x2 H-phosphonate building blocks and acyl, such as levulinoyl and substituted levulinoyl, groups.
When the H-phosphonate is a protected ribonucleoside or a protected oligoribonucleotide, the 2xe2x80x2-hydroxy function is advantageously protected by a suitable protecting group, for example an acid-labile acetal protecting group, particularly 1-(2-fluorophenyl)-4-methoxypiperidine-4-yl (Fpmp); and trialkylsilyl groups, often tri(C1-4-alkyl)silyl groups such as a tertiary butyl dimethylsilyl group. Alternatively, the ribonucleoside or oligoribonucleotide may be a 2xe2x80x2-O-alkyl, 2xe2x80x2-O-alkoxyalkyl or 2xe2x80x2-O-alkenyl derivative, commonly a C1-4 alkyl, C1-4 alkoxyC1-4alkyl or alkenyl derivative, in which case, the 2xe2x80x2 position does not need further protection.
Other H-phosphonates that may be employed in the process according to the present invention are derived from other polyfunctional alcohols, especially alkyl alcohols, and preferably diols or triols. Examples of alkyl diols include ethane-1,2-diol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400. Examples of alkyl triols include glycerol and butane triols. Commonly, only a single H-phosphonate function will be present, the remaining hydroxy groups being protected by suitable protecting groups, such as those disclosed hereinabove for the protection at the 5xe2x80x2 or 2xe2x80x2 positions of ribonucleosides.
The alcohol employed in the process of the present invention is commonly a protected nucleoside or oligonucleotide comprising a free hydroxy group, preferably a free 3xe2x80x2 or 5xe2x80x2 hydroxy group, and particularly preferably a 5xe2x80x2 hydroxy group.
When the alcohol is a protected nucleoside or a protected oligonucleotide, preferred nucleosides are deoxyribonucleosides and ribonucleosides and preferred oligonucleotides are oligodeoxyribonucleotides and oligoribonucleotides.
When the alcohol is a deoxyribonucleoside, ribonucleoside oligodeoxyribonucleotide or oligoribonucleotide derivative comprising a free 5xe2x80x2-hydroxy group, the 3xe2x80x2-hydroxy function is advantageously protected by a suitable protecting group. Examples of such protecting groups include acyl groups, commonly comprising up to 16 carbon atoms, such as those derived from gamma keto acids, such as levulinoyl groups and substituted levulinoyl groups. Substituted levulinoyl groups include particularly 5-halo-levulinoyl, such as 5,5,5-trifluorolevulinoyl and benzoylpropionyl groups. Other such protecting groups include fatty alkanoyl groups, including particularly linear or branched C6-16 alkanoyl groups, such as lauroyl groups; benzoyl and substituted benzoyl groups, such as alkyl, commonly C1-4 alkyl-, and halo, commonly chloro or fluoro, substituted benzoyl groups; and silyl ethers, such as alkyl, commonly C1-4 alkyl, and aryl, commonly phenyl, silyl ethers, particularly tertiary butyl dimethyl silyl and tertiary butyl diphenyl silyl groups.
When the alcohol is a protected deoxyribonucleoside, ribonucleoside, oligodeoxyribonucleotides or oligoribonucleotide comprising a free 3xe2x80x2-hydroxy group, the 5xe2x80x2-hydroxy function is advantageously protected by a suitable protecting group. Suitable protecting groups are those disclosed above for the protection of the 5xe2x80x2 hydroxy group of deoxyribonucleosides, ribonucleosides, oligodeoxyribonucleotides and oligoribonucleotide 3xe2x80x2 H phosphonates.
When the alcohol is a ribonucleoside or an oligoribonucleotide, the 2xe2x80x2-hydroxy function is advantageously protected by a suitable protecting group, such as an acetal, particularly 1-(2-fluorophenyl)4-methoxypiperidine4-yl (Fpmp); and trialkylsilyl groups, often tri(C1-4-alkyl)silyl groups such as a tertiary butyl dimethyl silyl group. Alternatively, the ribonucleoside or oligoribonucleotide may be a 2xe2x80x2-O-alkyl, 2xe2x80x2-O-alkoxyalkyl or 2xe2x80x2-O-alkenyl derivative, commonly a C1-4 alkyl, C1-4 alkoxyC1-4alkyl or alkenyl derivative, in which case, the 2xe2x80x2 position does not need further protection.
Other alcohols that may be employed in the process according to the present invention are non-saccharide polyols, especially alkyl polyols, and preferably diols or triols. Examples of alkyl diols include ethane-1,2-diol, and low molecular weight poly(ethylene glycols), such as those having a molecular weight of up to 400. Examples of alkyl triols include glycerol and butane triols. Commonly, only a single free hydroxy group will be present, the remaining hydroxy groups being protected by suitable protecting groups, such as those disclosed hereinabove for the protection at the 5xe2x80x2 or 2xe2x80x2 positions of ribonucleosides. However, more than one free hydroxy group may be present if it is desired to perform identical couplings on more than one hydroxy group.
When the H-phosphonate and the alcohol are both protected nucleosides or oligonucleotides, the invention provides an improved method for the stepwise and block synthesis in solution of oligodeoxyribonucleotides, oligoribonucleotides and analogues thereof, based on H-phosphonate coupling reactions. According to one preferred aspect of the present invention, protected nucleosides or oligonucleotides with a 3xe2x80x2-terminal H-phosphonate function and protected nucleosides or oligonucleotides with a 5xe2x80x2-terminal hydroxy function are coupled in the presence of a suitable coupling agent to form a protected dinucleoside or oligonucleotide H-phosphonate intermediate, wherein said intermediates undergo sulfur-transfer in situ in the presence of a suitable sulfur-transfer agent.
In addition to the presence of hydroxy protecting groups, bases present in nucleosides/nucleotides employed in present invention are also preferably protected where necessary by suitable protecting groups. Protecting groups employed are those known in the art for protecting such bases. For example, A and/or C can be protected by benzoyl, including substituted benzoyl, for example alkyl- or alkoxy-, often C1-4 alkyl- or C1-4alkoxy-, benzoyl; pivaloyl; and amidine, particularly dialkylaminomethylene, preferably di(C1-4-alkyl)aminomethylene such as dimethyl or dibutyl aminomethylene. G may be protected by a phenyl group, including substituted phenyl, for example 2,5-dichlorophenyl and also by an isobutyryl group. T and U generally do not require protection, but in certain embodiments may advantageously be protected, for example at O4 by a phenyl group, including substituted phenyl, for example 2,4-dimethylphenyl or at N3 by a pivaloyloxymethyl, benzoyl, alkyl or alkoxy substituted benzoyl, such as C1-4 alkyl- or C1-4 alkoxybenzoyl.
When the alcohol and/or H-phosphonate is a protected nucleoside or oligonucleotide having protected hydroxy groups, one of these protecting groups may be removed after carrying out the process of the first invention. Commonly, the protecting group removed is that on the 3xe2x80x2-hydroxy function. After the protecting group has been removed, the oligonucleotide thus formed may be converted into an H-phosphonate and may then proceed through further stepwise or block coupling and sulfur transfers according to the process of the present invention in the synthesis of a desired oligonucleotide sequence. The method may then proceed with steps to remove the protecting groups from the internucleotide linkages, the 3xe2x80x2 and the 5xe2x80x2-hydroxy groups and from the bases. Similar methodology may be applied to coupling 5xe2x80x2 H-phosphonates, wherein the protecting group removed is that on the 5xe2x80x2 hydroxy function.
In a particularly preferred embodiment, the invention provides a method comprising the coupling of a 5xe2x80x2-O-(4,4xe2x80x2-dimethoxytrityl)-2xe2x80x2-deoxyribonucleoside 3xe2x80x2-H-phosphonate or a protected oligodeoxyribonucleotide 3xe2x80x2-H-phosphonate and a component with a free 5xe2x80x2-hydroxy function in the presence of a suitable coupling agent and subsequent in situ sulfur transfer in the presence of a suitable sulfur-transfer agent.
In the process of the present invention, any suitable coupling agents and sulfur-transfer agents available in the prior art may be used.
Examples of suitable coupling agents include alkyl and aryl acid chlorides, alkane and arene sulfonyl chlorides, alkyl and aryl chloroformates, alkyl and aryl chlorosulfites and alkyl and aryl phosphorochloridates.
Examples of suitable alkyl acid chlorides which may be employed include C2 to C7 alkanoyl chlorides, particularly pivaloyl chloride. Examples of aryl acid chlorides which may be employed include substituted and unsubstituted benzoyl chlorides, such as C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted benzoyl chlorides. When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.
Examples of suitable alkanesulfonyl chlorides which may be employed include C2 to C7 alkanesulfonyl chlorides. Examples of arenesulfonyl chlorides which may be employed include substituted and unsubstituted benzenesulfonyl chlorides, such as C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted benzenesulfonyl chlorides. When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.
Examples of suitable alkyl chloroformates which may be employed include C2 to C7 alkyl chloroformates. Examples of aryl chloroformates which may be employed include substituted and unsubstituted phenyl chloroformates, such as C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted phenyl chloroformates. When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.
Examples of suitable alkyl chlorosulfites which may be employed include C2 to C7 alkyl chlorosulfites. Examples of aryl chlorosulfites which may be employed include substituted and unsubstituted phenyl chlorosulfites, such as C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted phenyl chlorosulfites. When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.
Examples of suitable alkyl phosphorochloridates which may be employed include di(C1 to C6 alkyl)phosphorochloridates. Examples of aryl phosphorochloridates which may be employed include substituted and unsubstituted diphenyl phosphorochloridates, such as C1-4 alkoxy, halo, particularly fluoro, chloro and bromo, and C1-4 alkyl, substituted diphenyl phosphorochloridates. When substituted, from 1 to 3 substituents are often present, particularly in the case of alkyl and halo substituents.
Further coupling agents that may be employed are the chloro-, bromo- and (benzotriazo-1-yloxy)-phosphonium and carbonium compounds disclosed by Wada et al, in J.A.C.S. 1997, 119, pp 12710-12721 (incorporated herein by reference).
Preferred coupling agents are diaryl phosphorochloridates, particularly those having the formula (ArO)2POCl wherein Ar is preferably phenyl, 2-chlorophenyl, 2,4,6-trichlorophenyl or 2,4,6-tribromophenyl.
The nature of the sulfur-transfer agent will depend on whether an oligonucleotide, a phosphorothioate analogue or a mixed oligonucleotide/oligonucleotide phosphorothioate is required. Sulfur transfer agents employed in the process of the present invention often have the general chemical formula:
Lxe2x80x94Sxe2x80x94A
wherein L represents a leaving group, and A represents an aryl group, a methyl or a substituted alkyl group or an alkenyl group. Commonly the leaving group is selected so as to comprise a nitrogen-sulfur bond. Examples of suitable leaving groups include morpholines such as morpholine-3,5-dione; imides such as phthalimides, succinimides and maleimides; indazoles, particularly indazoles with electron-withdrawing substituents such as 4-nitroindazoles; and triazoles.
Where a standard phosphodiester linkage is required in the final product, the sulfur transfer agent, the moiety A represents an aryl group, such as a phenyl or naphthyl group. Examples of suitable aryl groups include substituted and unsubstituted phenyl groups, particularly halophenyl and alkylphenyl groups, especially 4-halophenyl and 4-alkylphenyl, commonly 4-(C1-4 alkyl)phenyl groups, most preferably 4-chlorophenyl and p-tolyl groups. An example of a suitable class of standard phosphodiester-directing sulfur-transfer agent is an N-(arylsulfanyl)phthalimide (succinimide or other imide may also be used).
Where a phosphorothioate diester linkage is required in the final product, the moiety A represents a methyl, substituted alkyl or alkenyl group. Examples of suitable substituted alkyl groups include substituted methyl groups, particularly benzyl and substituted benzyl groups, such as alkyl-, commonly C1-4alkyl- and halo-, commonly chloro-, substituted benzyl groups, and substituted ethyl groups, especially ethyl groups substituted at the 2-position with an electron-withdrawing substituent such as 2-(4-nitrophenyl)ethyl and 2-cyanoethyl groups. Examples of suitable alkenyl groups are allyl and crotyl. Examples of a suitable class of phosphorothioate-directing sulfur-transfer agents are, for example, (2-cyanoethyl)sulfanyl derivatives such as 4-[(2-cyanoethyl)-sulfanyl]morpholine-3,5-dione or a corresponding reagent such as 3-(phthalimidosulfanyl)propanonitrile.
A suitable temperature for carrying out the coupling reaction and sulfur transfer is in the range of approximatelyxe2x88x9255xc2x0 C. to room temperature (commonly in the range of from 10 to 30xc2x0 C., for example approximately 20xc2x0 C.), and preferably from xe2x88x9240xc2x0 C. to 0xc2x0 C.
Organic solvents which can be employed in the process of the present invention include haloalkanes, particularly dichloromethane, esters, particularly alkyl esters such as ethyl acetate, and methyl or ethyl propionate, and basic, nucleophilic solvents such as pyridine. Preferred solvents for the coupling and sulfur transfer steps are pyridine, dichloromethane and mixtures thereof.
The mole ratio of H-phosphonate to alcohol in the process of the present invention is often selected to be in the range of from about 0.9:1 to 3:1, commonly from about 1:1 to about 2:1, and preferably from about 1.1:1 to about 1.5:1, such as about 1.2:1. However, where couplings on more than one free hydroxyl are taking place at the same time, the mole ratios will be increased proportionately. The mole ratio of coupling agent to alcohol is often selected to be in the range of from about 1:1 to about 10:1, commonly from about 1.5:1 to about 5:1 and preferably from about 2:1 to about 3:1. The mole ratio of sulfur transfer agent to alcohol is often selected to be in the range of from about 1:1 to about 10:1, commonly from about 1.5:1 to about 5:1 and preferably from about 2:1 to about 3:1.
In the process of the present invention, the H-phosphonate and the alcohol can be pre-mixed in solution, and the coupling agent added to this mixture. Alternatively, the H-phosphonate and the coupling agent can be pre-mixed, often in solution and then added to a solution of the alcohol, or the alcohol and the coupling agent may be mixed, commonly in solution, and then added to a solution of the H-phosphonate. In certain embodiments, the H-phosphonate, optionally in the form of a solution, can be added to a solution comprising a mixture of the alcohol and the coupling agent. After the coupling reaction is substantially complete, the sulfur transfer agent is then added to the solution the H-phosphonate diester produced in the coupling reaction. Reagent additions commonly take place continuously or incrementally over an addition period.
In the process of the present invention, it is possible to prepare oligonucleotides containing both phosphodiester and phosphorothioate diester internucleotide linkages in the same molecule by selection of appropriate sulfur transfer agents, particularly when the process is carried out in a stepwise manner.
As stated previously, the method of the invention can be used in the synthesis of RNA, 2xe2x80x2-O-alkyl-RNA, 2xe2x80x2-O-alkoxyalkyl-RNA and 2xe2x80x2-O-alkenyl-RNA sequences. 2xe2x80x2-O(Fpmp)-5xe2x80x2-O-(4,4-dimethoxytrityl)-ribonucleoside 3xe2x80x2-H-phosphonates 1 and 2xe2x80x2-O-(alkyl, alkoxyalkyl or alkenyl)-5xe2x80x2-O-(4,4-dimethoxytrityl)-ribonucleoside 3xe2x80x2-H-phosphonates 2a-c may be prepared, for example, from the corresponding nucleoside building blocks, ammonium p-cresyl H-phosphonate and pivaloyl chloride. 
The same protocols are used as in the synthesis of DNA and DNA phosphorothioate sequences (Schemes 2-4). Following the standard unblocking procedure (Scheme 2, steps v and vi), the Fpmp protecting groups are removed under mild conditions of acidic hydrolysis that lead to no detectable cleavage or migration of the internucleotide linkages (Capaldi, D. C.; Reese, C. B. Nucleic Acids Res. 1994, 22, 2209-2216). For chemotherapeutically useful ribozyme sequences, relatively large scale RNA synthesis in solution is a matter of considerable practical importance. The incorporation of 2xe2x80x2-O-alkyl, 2xe2x80x2-O-substituted alkyl and 2xe2x80x2-O-alkenyl [especially 2xe2x80x2-O-methyl, 2xe2x80x2-O-allyl and 2xe2x80x2-O-(2-methoxyethyl)]-ribonucleosides (Sproat, B. S. in xe2x80x98Methods in Molecular Biology, Vol. 20. Protocols for Oligonucleotides and Analogsxe2x80x99, Agrawal, S., Ed., Humana Press, Totowa, 1993) into oligonucleotides is currently a matter of much importance as these modifications confer both resistance to nuclease digestion and good hybridisation properties on the resulting oligomers.
The sulfur transfer step is carried out on the product of the H-phosphonate coupling in situ, ie without separation and purification of the intermediate produced by the coupling reaction. Preferably, the sulfur transfer agent is added to the stirred mixture resulting from the coupling reaction.
In addition to the fact that it is carried out in homogenous solution, the present coupling procedure differs from that followed in the H-phosphonate approach to solid phase synthesis (Froehler et al., Methods in Molecular Biology, 1993) in at least two other important respects. First, it may be carried out at a very low temperature. Side reactions which can accompany H-phosphonate coupling (Kuyl-Yeheskiely et al, Rec. Trav. Chim., 1986, 105, 505-506) can thereby be avoided even when di-(2-chlorophenyl)phosphorodichloridate rather than pivaloyl chloride (Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett., 1986, 27, 469-472) is used as the coupling reagent. Secondly, sulfur transfer is carried out after each coupling step rather than just once following the assembly of the whole oligomer sequence.
Protecting groups can be removed using methods known in the art for the particular protecting group and function. For example, transient protecting groups, particularly gamma keto acids such as levulinoyl-type protecting groups, can be removed by treatment with hydrazine, for example, buffered hydrazine, such as the treatment with hydrazine under very mild conditions disclosed by van Boom. J. H.; Burgers, P. M. J. Tetrahedron Lett., 1976, 4875-4878. The resulting partially-protected oligonucleotides with free 3xe2x80x2-hydroxy functions may then be converted into the corresponding H-phosphonates which are intermediates which can be employed for the block synthesis of oligonucleotides and their phosphorothioate analogues.
When deprotecting the desired product once this has been produced, protecting groups on the phosphorus which produce phosphorothioate triester linkages are commonly removed first. For example, a cyanoethyl group can be removed by treatment with a strongly basic amine such as DABCO, 1,5-diazabicylo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine.
Phenyl and substituted phenyl groups on the phosphorothioate internucleotide linkages and on the base residues can be removed by oximate treatment, for example with the conjugate base of an aldoxime, preferably that of E-2-nitrobenzaldoxime or pyridine-2-carboxaldoxime (Reese et al, Nucleic Acids Res. 1981). Kamimura, T. et al in J. Am. Chem. Soc., 1984, 106 4552-4557 and Sekine, M. Et al, Tetrahedron, 1985, 41, 5279-5288 in an approach to oligonucleotide,synthesis by the phosphotriester approach in solution, based on S-phenyl phosphorothioate intermediates; and van Boom and his co-workers in an approach to oligonucleotide synthesis, based on S-(4-methylphenyl)phosphorothioate intermediates (Wreesman, C. T. J. Et al, Tetrahedron Lett., 1985, 26, 933-936) have all demonstrated that unblocking S-phenylphosphorothioates with oximate ions (using the method of Reese et al., 1978; Reese, C. B,; Zard, L. Nucleic Acids Res., 1981, 9, 4611-4626) led to natural phosphodiester internucleotide linkages. In the present invention, the unblocking of S-(4-chlorophenyl)-protected phosphorothioates with the conjugate base of E-2-nitrobenzaldoxime proceeds smoothly and with no detectable internucleotide cleavage.
Other base protecting groups, for example benzoyl, pivaloyl and amidine groups can be removed by treatment with concentrated aqueous ammonia.
Trityl groups present can be removed by treatment with acid. With regard to the overall unblocking strategy in oligodeoxyribonucleotide synthesis, another important consideration of the present invention, is that the removal of trityl, often a 5xe2x80x2-terminal DMTr, protecting group (xe2x80x98detritylationxe2x80x99) should proceed without concomitant depurination, especially of any 6-N-acyl-2xe2x80x2-deoxyadenosine residues. According to an embodiment of the invention, the present inventors have found that such depurination, which perhaps is difficult completely to avoid in solid phase synthesis, can be totally suppressed by effecting xe2x80x98detritylationxe2x80x99 with a dilute solution of hydrogen chloride at low temperature, particularly ca. 0.45 M hydrogen chloride in dioxanexe2x80x94dichloromethane (1:8 v/v) solution atxe2x88x9250xc2x0 C. Under these reaction conditions, xe2x80x98detritylationxe2x80x99 can be completed rapidly, and in certain cases after 5 minutes or less. For example, when 6-N-benzoyl-5xe2x80x2-O-(4,4xe2x80x2-dimethoxytrityl)-2xe2x80x2-deoxyadenosine was treated with hydrogen chloride in dioxanexe2x80x94dichloromethane under such conditions, xe2x80x98detritylationxe2x80x99 was complete after 2 min, but no depurination was detected even after 4 hours.
Silyl protecting groups may be removed by fluoride treatment, for example with a solution of a tetraalkyl ammonium fluoride salt such as tetrabutyl ammonium fluoride.
Fpmp protecting groups may be removed by acidic hydrolysis under mild conditions.
This new approach to the synthesis of oligonucleotides in solution is suitable for the preparation of sequences with (a) solely phosphodiester, (b) solely phosphorothioate diester and (c) a combination of both phosphodiester and phosphorothioate diester internucleotide linkages.
The invention also relates to the development of block coupling (as illustrated for example in Scheme 4b). In this respect, the examples provide an illustration of the synthesis of d[Tp(s)Tp(s)Gp(s)Gp(s)Gp(s)Gp(s)Tp(s)T] (ISIS 5320 Ravikuma, V. T.; Cherovallath, Z. S. Nucleosides and Nucleosides 1996, 15, 1149-1155), an octadeoxyribonucleoside heptaphosphorothioate, from tetramer blocks. This oligonucleotide analogue has properties as an anti-HIV agent. Other proposed block synthesis targets include sequences with therapeutic effects, for example, inhibitors of human thrombin and anti-HIV agents. The method of the invention furthermore can be used in the synthesis of larger sequences.
It will be apparent that when the process of the present invention is applied to block synthesis, a number of alternative strategies are available in terms of the route to the desired product. These will depend on the nature of the desired product. For example, an octamer may be prepared by the preparation of dimers, coupled to produce tetramers, which are then coupled to produce the desired octamer. Alternatively, a dimer and a trimer may be coupled to produce a pentamer, which can be coupled with a further trimer to produce the desired octamer. The choice of strategy is at the discretion of the user. However, the common feature of such block coupling is that an oligomer H-phosphonate comprising two or more units is coupled with an oligomer alcohol also comprising two or more units. Most commonly oligonucleotide 3xe2x80x2-H-phosphonates are coupled with oligonucleotides having free 5xe2x80x2-hydroxy functions.
The process of the present invention can also be employed to prepare cyclic oligonucleotides, especially cyclic oligodeoxyribonucleotides and cyclic ribonucleotides. In the preparation of cyclic oligonucleotides, an oligonucleotides comprising an H-phosphonate function, often a 3xe2x80x2 or 5xe2x80x2 H-phosphonate is prepared, and a free hydroxy function is introduced by appropriate deprotection. The position of the free hydroxy function is usually selected to correspond to the H-phosphonate, for example a 5xe2x80x2 hydroxy function would be coupled with a 3xe2x80x2 H-phosphonate, and a 3xe2x80x2 hydroxy function would be coupled with a 5xe2x80x2 H-phosphonate. The hydroxy and the H-phosphonate functions can then be coupled intramolecularly in solution in the presence of a coupling agent and this reaction is followed by in situ sulfur transfer.
According to a further aspect of the present invention, there is provided novel oligomer H-phosphonates having the general chemical formula: 
wherein
each B independently is a base selected from A, G, T, C or U;
each Q independently is H or ORxe2x80x2 wherein Rxe2x80x2 is alkyl, substituted alkyl, alkenyl or a protecting group;
each R independently is an aryl, methyl, substituted alkyl or alkenyl group;
W is H, a protecting group or an H-phosphonate group of formula 
xe2x80x83in which
M+ is a monovalent cation;
each X independently represent O or S;
each Y independently represents O or S;
Z is H, a protecting group or an H-phosphonate group of formula 
xe2x80x83in which
M+ is a monovalent cation; and
n is an integer and is at least 2;
provided that when W is H or a protecting group, that Z is an H-phosphonate group, and that when Z is H or a protecting group, that W is an H-phosphonate group.
Also provided are novel oligomer H-phosphonates having the general chemical formula: 
wherein
each B independently is a base selected from A, G, T, C or U;
each Q independently is H or ORxe2x80x2 wherein Rxe2x80x2 is alkyl, substituted alkyl, alkenyl or a protecting group;
each R independently is an aryl, methyl, substituted alkyl or alkenyl group;
W is H, a protecting group or an H-phosphonate group of formula 
xe2x80x83in which
M+ is a monovalent cation;
each X independently represent O or S;
each Y represents S;
Z is H, a protecting group or an H-phosphonate group of formula 
xe2x80x83in which
M+ is a monovalent cation; and
n is a positive integer;
provided that when W is H or a protecting group, that Z is an H-phosphonate group, and that
when Z is H or a protecting group, that W is an H-phosphonate group.
Preferably, only one of W or Z is an H-phosphonate group, commonly only Z being an H-phosphonate group.
When W or Z represents a protecting group, the protecting group may be one of those disclosed above for protecting the 3xe2x80x2 or 5xe2x80x2 positions respectively. When W is a protecting group, the protecting group is a trityl group, particularly a dimethoxytrityl group. When Z is a protecting group, the protecting group is a trityl group, particularly a dimethoxytrityl group, or an acyl, preferably a levulinoyl group.
The bases A, G and C represented by B are preferably protected, and bases T and U may be protected. Suitable protecting groups include those described hereinabove for the protection of bases in the process according to the first aspect of the present invention.
When Q represents a group of ORxe2x80x2, and Rxe2x80x2 is alkenyl, the alkenyl group is often a C1-4 alkenyl group, especially allyl or crotyl group. When Rxe2x80x2 represents alkyl, the alkyl is preferably a C1-4 alkyl group. When Rxe2x80x2 represents substituted alkyl, the substituted alkyl group includes alkoxyalkyl groups, especially C1-4 alkyoxyC1-4 alkyl groups such as methoxyethyl groups. When Rxe2x80x2 represents a protecting group, the protecting group is commonly an acid-labile acetal protecting group, particularly 1-(2-fluorophenyl)4-methoxypiperidine4-yl (Fpmp) or a trialkylsilyl groups, often a tri(C1-4-alkyl)silyl group such as a tertiary butyl dimethylsilyl group.
Preferably, X represents O.
In many embodiments, Y represents S and each R represents the methyl, substituted alkyl, alkenyl or aryl group remaining from the sulfur transfer agent(s) employed in the process of the present invention. Preferably, each R independently represents a methyl group; a substituted methyl group, particularly a benzyl or substituted benzyl group, such as an alkyl-, commonly C1-4alkyl- or halo-, commonly chloro-, substituted benzyl group; a substituted ethyl group, especially an ethyl group substituted at the 2-position with an electron-withdrawing substituent such as a 2-(4-nitrophenyl)ethyl or a 2-cyanoethyl group; a C1-4 alkenyl group, preferably an allyl and crotyl group; or a substituted or unsubstituted phenyl group, particularly a halophenyl or alkylphenyl group, especially 4-halophenyl group or a 4-alkylphenyl, commonly a 4-(C1-4 alkyl)phenyl group, and most preferably a 4-chlorophenyl or a p-tolyl group.
M+preferably represents a trialkyl ammonium ion, such as a tri(C1-4alkylammonium) ion, and preferably a triethylammonium ion.
n may be 1 up to any number depending on the oligonucleotide which is intended to be synthesised, particularly up to about 20. Preferably n is 1 to 16, and especially 1 to 9. H-phosphonate wherein n represents 1, 2 or 3 can be employed when it is desired to add small blocks of nucleotide, with correspondingly larger values of n, for example 5, 6 or 7 or more being employed if larger blocks of oligonucleotide are desired to be coupled.
The H-phosphonates according to the present invention are commonly in the form of solutions, preferably those employed in the process of the first aspect of the present invention.
These H-phosphonates are also useful intermediates in the block synthesis of oligonucleotides and oligonucleotide phosphorothioates. As indicated above, block coupling is much more feasible in solution phase than in solid phase synthesis.
The oligonucleotide H-phosphonates can be prepared using general methods known in the art for the synthesis of nucleoside H-phosphonates. Accordingly, in a further aspect of the present invention, there is provided a process for the production of an oligonucleotide H-phosphonate wherein an oligonucleotide comprising a free hydroxy function, preferably a 3xe2x80x2 or 5xe2x80x2 hydroxy function, is reacted with an alkyl or aryl H-phosphonate salt in the presence of an activator.
Preferably, the oligonucleotide is a protected oligonucleotide, and most preferably a protected oligodeoxynucleotide or a protected oligoribonucleotide. The H-phosphonate salt is often an ammonium salt, including alkyl, aryl and mixed alkyl and aryl ammonium salts. Preferably, the ammonium salt is an (NH4)+ or a tri(C1-4alkyl) ammonium salt. Examples of alkyl groups which may be present in the H-phosphonate are C1-4 alkyl, especially C2-4 alkyl, groups substituted with strongly electron withdrawing groups, particularly halo, and preferably fluoro groups, such as 2,2,2-trifluoroethyl and 1,1,1,3,3,3-hexafluoropropan-2-yl groups. Examples of aryl groups which may be present include phenyl and substituted phenyl, particularly alkylphenyl, commonly C1-4 alkylphenyl and halophenyl, commonly chlorophenyl groups. Preferably, a substituted phenyl group is a 4-substituted phenyl group. Particularly preferred H-phosphonates are ammonium and triethylammonium p-cresyl H-phosphonates. Activators which may be employed include those compounds disclosed herein for use as coupling agents, and particularly diaryl phosphorochloridates and alkyl and cycloalkyl acid chlorides, such as 1-adamantanecarbonyl chloride, and preferably pivaloyl chloride. The production of H-phosphonates preferably takes place in the presence of a solvent, often those solvents disclosed for use in the process of the first aspect of the present invention, preferably pyridine, dichloromethane and mixtures thereof.
One advantage of the present invention for the synthesis of solely phosphorothioate diesters is that, provided care is taken to avoid desulfurisation during the unblocking steps [particularly during heating with aqueous ammonia (for example Scheme 3, step viii(a))], the synthesis of oligonucleotide phosphorothioates should not lead to products that are contaminated with standard phosphodiester internucleotide linkages. In the case of solid phase oligonucleotide phosphorothioate synthesis, incomplete sulfur transfer in each synthetic cycle usually leads to a residual phosphodiester contamination (Zon, G.; Stec, W. J. in xe2x80x98Oligonucleotides and Analogs. A Practical Approachxe2x80x99, Eckstein, F., Ed., IRL Press, Oxford, 1991, pp. 87-108).
The solution synthesis as proposed by the present invention has another enormous advantage over solid-phase synthesis in that the possibility exists of controlling the selectivity of reactions by working at low or even at very low temperatures. This advantage extends to the detritylation step (Scheme 3, step i) which can proceed rapidly and quantitatively below 0xc2x0 C. without detectable depurination. After the detritylation step, a relatively quick and efficient purification can be effected by what has previously been described as the xe2x80x98filtrationxe2x80x99 approach (Chaudhuri, B,; Reese, C. B.; Weclawek, K. Tetrahedron Lett. 1984, 25, 4037-4040). This depends on the fact that phosphotriester (and phosphorothioate triester) intermediates, but not any remaining detritylated charged monomers, are very rapidly eluted from short columns of silica gel by THF-pyridine mixtures.
The method according to the invention will now be illustrated with reference to the following examples which are not intended to be limiting:
In the Examples, it should be noted, that where nucleoside residues and internucleotide linkages are italicised, this indicates that they are protected in some way. In the present context, A, C, G, and T represent 2xe2x80x2-deoxyadenosine protected on N-6 with a benzoyl group, 2xe2x80x2-deoxycytidine protected on N-4 with a benzoyl group, 2xe2x80x2-deoxyguanosine protected on N-2 and on O-6 with isobutyryl and 2,5-dichlorophenyl groups and unprotected thymine. For example, as indicated in scheme 3, p(s) and p(sxe2x80x2) represent S-(2-cyanoethyl) and S-(4-chlorophenyl)phosphorothioates, respectively, and p(H), which is not protected and therefore not italicised, represents an H-phosphonate monoester if it is placed at the end of a sequence or attached to a monomer but otherwise it represents an H-phosphonate diester.